Methods for identifying reactive functional cysteines in nuclear proteins

Abstract

The disclosure relates to compositions and methods for identifying reactive cysteines on nuclear proteins from cell samples. Further wherein the method includes contacting live cells, dead cells, cell lysates, nuclei, or a nuclear fraction obtained from cells with an electrophilic probe, and performing mass spectrometry analysis of proteins present in the cytoplasmic fraction.

I/We claim: 
     
         1 . A method for identifying reactive cysteine residues present in cellular proteins, the method comprising:
 (a) contacting live cells, dead cells, cell lysates, nuclei, or a nuclear fraction obtained from cells with an electrophilic probe;   (b) optionally harvesting and lysing the live cells or the dead cells of (a);   (c) optionally separating nuclei from the lysed cells of (b), and optionally collecting the supernatant from the separated nuclei as a cytoplasmic fraction;   (d) optionally extracting nuclear proteins by resuspending the nuclei of (a) or the separated nuclei of (c) in an isotonic buffer comprising salt (and optionally not comprising polycations);   (e) optionally separating insoluble chromatin and collecting the supernatant from the separated insoluble chromatin as a nuclear fraction; and   (f) performing mass spectrometry analysis of proteins present in the cytoplasmic fraction of (c) and/or the nuclear fraction of (a) or (e) to identify reactive cysteines modified by the electrophilic probe,   thereby identifying reactive cysteines present in cellular proteins.   
     
     
         2 . A method for identifying reactive cysteine residues present in cellular proteins, the method comprising:
 (a) contacting live cells with an electrophilic probe;   (b) harvesting and lysing the cells of (a);   (c) separating nuclei from the lysed cells of (b) and collecting the supernatant from the separated nuclei as a cytoplasmic fraction;   (d) resuspending the separated nuclei of (c) in an isotonic buffer comprising salt (and optionally not comprising polycations);   (e) separating insoluble chromatin from the resuspended nuclei of (d) and collecting the supernatant from the separated insoluble chromatin as a nuclear fraction; and   (f) performing mass spectrometry analysis of proteins present in the cytoplasmic fraction and/or nuclear fraction to identify reactive cysteines modified by the electrophilic probe,   thereby identifying reactive cysteines present in cellular proteins.   
     
     
         3 . A method for identifying reactive cysteine residues present in cellular proteins, the method comprising:
 (a) contacting live cells, dead cells, cell lysates, nuclei, or a nuclear fraction obtained from cells with an electrophilic probe;   (b) optionally harvesting and lysing the live cells or the dead cells of (a);   (c) optionally separating nuclei from the lysed cells of (b), and optionally collecting the supernatant from the separated nuclei as a cytoplasmic fraction, wherein the cytoplasmic fraction is enriched for cytosolic proteins;   (d) optionally extracting nucleoplasm associated proteins, euchromatin associated proteins, and/or heterochromatin associated proteins as follows:
 (i) resuspending the nuclei of (a) or the separated nuclei of (c) in an isotonic buffer comprising salt (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a nuclear fraction, wherein the nuclear fraction is enriched for nucleoplasm associated proteins; 
 (ii) resuspending the insoluble chromatin of (i) in a low salt buffer comprising an increased salt concentration as compared to the isotonic buffer (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as an euchromatin fraction, wherein the euchromatin fraction is enriched for euchromatin associated proteins; and/or 
 (iii) resuspending the insoluble chromatin of (i) or (ii) in a high salt buffer comprising an increased salt concentration as compared to the low salt buffer (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a heterochromatin fraction, wherein the heterochromatin fraction is enriched for heterochromatin associated proteins; and 
   (e) performing mass spectrometry analysis of proteins present in one or more of the cytoplasmic fraction, nuclear fraction, euchromatin fraction, and heterochromatin fraction to identify reactive cysteines modified by the electrophilic probe,   thereby identifying reactive cysteines present in cellular proteins.   
     
     
         4 . A method for identifying reactive cysteine residues present in cellular proteins, the method comprising:
 (a) contacting live cells with an electrophilic probe;   (b) harvesting and lysing the cells of (a);   (c) separating nuclei from the lysed cells of (b) and collecting the supernatant from the separated nuclei as a cytoplasmic fraction;   (d) extracting one or more nuclear protein fractions as follows:
 (i) resuspending the separated nuclei of (c) in an isotonic buffer comprising salt (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a nuclear fraction; 
 (ii) resuspending the insoluble chromatin of (i) in a low salt buffer comprising an increased salt concentration as compared to the isotonic buffer (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as an euchromatin fraction; and/or 
 (iii) resuspending the insoluble chromatin of (i) or (ii) in a high salt buffer comprising an increased salt concentration as compared to the low salt buffer (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a heterochromatin fraction; and 
   (e) performing mass spectrometry analysis of proteins present in one or more of the cytoplasmic fraction, nuclear fraction, euchromatin fraction, and heterochromatin fraction to identify reactive cysteines modified by the electrophilic probe,   thereby identifying reactive cysteines present in cellular proteins.   
     
     
         5 . A method for identifying reactive cysteine residues present in cellular proteins, the method comprising:
 (a) contacting live cells with an electrophilic probe;   (b) harvesting and lysing the cells of (a);   (c) separating nuclei from the lysed cells of (b) and collecting the supernatant from the separated nuclei as a cytoplasmic fraction;   (d) extracting one or more nuclear protein fractions as follows:
 (i) resuspending the separated nuclei of (c) in an isotonic buffer comprising salt (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a nuclear fraction; and 
 (ii) resuspending the insoluble chromatin of (i) in a high salt buffer comprising an increased salt concentration as compared to the isotonic buffer (and optionally not comprising polycations), separating insoluble chromatin, and collecting the supernatant as a chromatin fraction; and 
   (e) performing mass spectrometry analysis of proteins present in one or more of the cytoplasmic fraction, nuclear fraction, and chromatin fraction to identify reactive cysteines modified by the electrophilic probe,   thereby identifying reactive cysteines present in cellular proteins.   
     
     
         6 . The method of any one of  claims 1-5 , further comprising homogenizing, washing, and/or pelleting the cells before lysing the cells. 
     
     
         7 . The method of any one of  claims 1-6 , wherein the cells are lysed by contacting them with a first suspension buffer comprising a detergent, optionally wherein the detergent comprises NP40 or Triton X-100, and optionally wherein the detergent is present at a concentration of from about 0.1% to about 4%. 
     
     
         8 . The method of any one of  claims 1-7 , further comprising incubating the nuclei and/or insoluble chromatin in one or more buffer(s) before separating the nuclei or insoluble chromatin from the supernatant. 
     
     
         9 . The method of  claim 8 , wherein the nuclei are incubated at a temperature of about 4° C. 
     
     
         10 . The method of  claim 8 , wherein the nuclei are incubated for a period of about 30 minutes. 
     
     
         11 . The method of any one of  claims 1-10 , wherein the salt comprises sodium chloride (NaCl). 
     
     
         12 . The method of  claim 11 , wherein the isotonic buffer comprises NaCl at a concentration of about at a concentration of about 10 mM to about 20 mM, the low salt buffer comprises NaCl at a concentration of about 100 mM to about 400 mM, and/or the high salt buffer comprises NaCl at a concentration of about 400 mM to about 800 mM. 
     
     
         13 . The method of  claim 12 , wherein the isotonic buffer comprises NaCl at a concentration of about 15 mM, the low salt buffer comprises NaCl at a concentration of about 250 mM, and/or the high salt buffer comprises NaCl at a concentration of about 600 mM. 
     
     
         14 . The method of any one of  claims 1-13 , further comprising quenching the nuclei, optionally after step (c). 
     
     
         15 . The method of  claim 14 , wherein the nuclei are quenched with ethylenediaminetetraacetic acid (EDTA). 
     
     
         16 . The method of  claim 15 , wherein the EDTA is present at a concentration of from 0.1 mM to 10 mM. 
     
     
         17 . The method of any one of  claims 1-16 , wherein the method further comprises treating the nuclei with a nuclease. 
     
     
         18 . The method of  claim 17 , wherein the nuclei are treated with the nuclease at a temperature of about 37° C. and/or for a period of about 5 minutes. 
     
     
         19 . The method of any one of  claims 1-18 , further comprising adding a surfactant to the one or more supernatants and/or the insoluble chromatin. 
     
     
         20 . A method for determining subcellular location of one or more cellular proteins, comprising:
 (a) performing the method of any one of claims  1 - 19 ; and   (b) determining the subcellular location of the one or more cellular proteins,   wherein the subcellular location is selected from the group consisting of cytoplasmic, nuclear, nucleoplasm associated, euchromatin associated, and heterochromatin associated.   
     
     
         21 . A method for determining if a condition alters subcellular location of one or more cellular proteins, comprising:
 (a) performing the method of any one of claims  1 - 19  on cells subjected to a first condition; and   (b) performing the method of any one of claims  1 - 19  on cells subjected to a second condition; and   (c) determining the subcellular location of the one or more cellular protein following step (a) and step (b), wherein the subcellular location is selected from the group consisting of cytoplasmic, nucleoplasm associated, euchromatin associated, and heterochromatin associated,   thereby determining whether the first or second condition alters the subcellular location of the one or more cellular proteins.   
     
     
         22 . The method of  claim 21 , wherein the first and second conditions are selected from:
 (a) a first and second environmental condition;   (b) a first and second cell state;   (c) before and after treatment with one or more candidate therapeutic agent;   (d) before and after treatment with a small molecule degradation compound;   (e) before and after a gene modification of the cells, optionally by genome editing;   (f) before and after expression of a transgene in the cells;   (g) a first and second thermal condition; and   (h) healthy cells versus diseased or injured cells.   
     
     
         23 . The method of any one of  claims 20-22 , wherein the cellular proteins are transcription factors. 
     
     
         24 . The method of any one of  claims 1-23 , wherein the electrophilic probe comprises a reactive group selected from the group consisting of iodoacetamides, chloroacetamides, epoxides, acrylamides, acyl halogens, sulfonate esters, acyloxymethyl ketones, vinylsulfonamides, propynamides, and malemides 
     
     
         25 . The method of any one of  claims 1-24 , wherein the electrophilic probe is an acrylamide or a derivative thereof, an iodoacetamide or a derivative thereof, a chloroacetamide or a derivative thereof, a propynamide or a derivative thereof, or a malemide or a derivative thereof. 
     
     
         26 . The method of  claim 25 , wherein the electrophilic probe is n-methyl iodoacetamide (NM-IAA). 
     
     
         27 . The method of any one of  claims 1-26 , wherein the cells comprise cancer cells. 
     
     
         28 . The method of any one of  claims 1-26 , wherein the cells comprise immune cells.